Colorectal cancer (CRC) is a genetic disease governed by clonal evolution1. Genotyping CRC tissue is employed for therapeutic purposes but this approach has significant limitations. A tissue sample represents a single snapshot in time, is subjected to selection bias due to tumor heterogeneity, and can be difficult to obtain. We exploited circulating DNA (ctDNA) to genotype colorectal tumors and track clonal evolution during therapies with the anti-EGFR antibodies cetuximab or panitumumab. We identified genomic alterations in KRAS, NRAS, MET, ERBB2, FLT3, EGFR and MAP2K1 in ctDNA of patients with primary or acquired resistance to EGFR blockade. Mutant RAS clones, which rise in blood during EGFR blockade, decline upon withdrawal of anti-EGFR antibodies indicating that clonal evolution continues beyond clinical progression. Pharmacogenomic analysis of CRC cells, which had acquired resistance to cetuximab, reveals that upon antibody withdrawal KRAS clones decay, while the population regains drug sensitivity. ctDNA profiles of patients who benefit from multiple challenging with anti-EGFR antibodies exhibit pulsatile levels of mutant KRAS. These results reveal that the CRC genome adapts dynamically to intermittent drug schedules and provide a molecular explanation for the efficacy of re-challenge therapies based on EGFR blockade.

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These data demonstrate that primary tumor location is an important prognostic factor in previously untreated mCRC. Given the consistency across an exploratory set and two confirmatory phase III studies, side of tumor origin should be considered for stratification in randomized trials.

ALK, ROS1, and NTRK rearrangements define a new rare subtype of mCRC with extremely poor prognosis. Primary tumor site, MSI-high, and RAS and BRAF wild-type status may help to identify patients bearing these alterations. While sensitivity to available treatments is limited, targeted strategies inhibiting ALK, ROS, and TrkA-B-C provided encouraging results.